1. Field of the Invention
This invention pertains to an assay format for the detection of the presence of luciferase, as a reporting molecule or target molecule, in an assay. The assay finds particular value in assays for the detection of luciferase present in a biological sample as the expression product of a luciferase gene transfected into a host cell as a reporter gene. Additionally, compositions useful in the assay method, and kits for performing the assay, are addressed.
2. Background of Prior Art
Chemiluminescent assays using luciferase as an enzyme to indicate the presence of ATP in a biological system or sample are widely described in the literature. Luciferase catalyzes chemiluminescent light release according to the following reaction system: ##STR1##
Although a variety of luciferase enzymes have been identified, and all that catalyze a similar reaction utilizing ATP may be used in conjunction with the invention of this application, the best characterized luciferase is firefly luciferase, the enzyme responsible for the characteristic "flash" of this particular beetle (P. pyralis). This luciferase is a 61-62 kD monomeric enzyme which catalyzes the light emission described above with a quantum yield of 0.88. The light naturally emitted at pH 7.8 is a green-yellow light (about 562 nm).
Luciferase assays for the quantitiation of luciferase enzyme are described in U.S. Pat. Nos. 5,652,289 and 5,283,179, Wood. A well known difficulty encountered in developing luciferase assays is the enzyme kinetics which produces a very rapid peak intensity, i.e., the "flash of light", coupled with a rapid decay to a very low level, is discussed extensively in these patents. The patents also address prior art attempts to solve this problem, including the addition of coenzyme A (CoA) and/or a sulfhydryl compound such as dithiothreitol (DTT) to support greater overall light release.
A wide variety of literature has been developed concerning luciferase assays, and methods to increase both peak light intensity and total light emission. Among the various alternatives explored is the selection of specific solvents, Kricka, et al., Arch. Biochemistry and Biophysics 217, 674-681 (1982), other SH compounds such as N-acetylcystein, Lundin, Bioluminescence and Chemiluminescence, and the addition of sodium arsenate and other salts, DeLuca et al., Analytical Biochemistry 95, 194-198 (1978).
Specific assays employing these formulations to detect target molecules at as low as picomolar concentrations are described in Olsson, et al., Journal of Applied Biochemistry 5, 437-445 (1983), Kimmich, et al., Analytical Biochemistry 69, 187-206 (1975), Brovko, et al., Analytical Biochemistry 220, 410-414 (1994), Momsen, Biochemical and Biophysical Research Communications 84, 816-822 (1978) and Holmsen, et al., Analytical Biochemistry 46, 489-501 (1972). Many of these references describe the role of myokinase, also known as adenylate kinase, for measurement of an adenine nucleotide with a luciferase reaction, (see, in particular, Brovko, Momsen, Olsen and Holmsen, supra). Myokinase is a ubiquitous enzyme found in both prokaryotic and eukaryotic cells, with a molecular weight in its monomeric form of about 21 kDa. An important feature of myokinase is its high substrate specificity to adenine nucleotides, being reactive with ADP, AMP and ATP. Myokinase catalyzes a bidirectional reaction: ##STR2##
Just as ATP and Mg.sup.++ are essential components to drive the luciferase chemiluminescent reaction forward, AMP is a known inhibitor of the luciferase light-yielding reaction.
U.S. Pat. No. 5,618,682, Scheirer, addresses a luciferase assay based on the luciferase-luciferin reaction which is described as providing light emission over a period of up to 8 hours. In this assay, a sample, which is suspected of containing luciferase, is combined with the luciferase substrate luciferin, ATP, AMP, and other co-factors for promoting catalytic activity. Essential to this patent is the combination of both ATP, to support the luciferase-catalyzed reaction, and AMP, to retard the enzyme kinetics, so as to produce a sustained light emission.
As mentioned by Scheirer, luciferase has become important as a reporter molecule outside of the traditional environment of measurement of ATP, as an indicator of ATP synthesis, metabolism, or as an indicator for the presence of bacterial cells. A number of genes for luciferase have been identified and been demonstrated to be effective, when transfected, in expression in heterologous hosts. Thus, the luciferase gene has become an important reporter gene, since detection of the presence of luciferase in a recombinant cell candidate can be used in a wide variety of environments, provided light emission intensity and duration can be supported so as to enable for facile detection, and importantly, automated detection.
Accordingly, it remains an object of those of ordinary skill in the art to develop a luciferase assay which provides, simultaneously, sufficient sensitivity and light intensity, over a sustained period of time, to permit automated chemiluminescent detection of the presence of luciferase in a sample, particularly as a reporter gene in recombinant cell assays.